PROJECT SUMMARY Immune dysfunction plays a critical role in the development of the lethal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), which results from infection of oligodendrocytes and astrocytes in the brain by human neurotropic Polyomavirus JC (JCV). JCV is ubiquitous in the human population worldwide and after primary infection early in life, it sustains an asymptomatic persistent infection. Later, JCV may reactivate under conditions of immunosuppression to cause PML. At present, there is no accepted effective therapy for PML, which is often fatal within a few months. Interplay between viral and host factors and the signaling pathways that regulate JCV productive replication remain poorly defined. Recently, we found that JCV infection of glial cells causes significant cellular DNA damage. This damage is associated with induction of the cellular DNA damage response (DDR), which involves activation of phospho-ATM (ataxia telangiectasia-mutated protein), gamma-H2AX phosphorylation and an increased expression level of the DNA repair protein Rad51. Activated ATM has an important role in coordinating the molecular events involved in DNA damage signaling and repair and the activation of nuclear factor kappa-B (NF-KB) by a novel NF-KB mechanism known as nucleus to cytoplasm or ?inside-out? NF-KB signaling. Importantly, we found that Rad51 complexes with NF-KB p65 subunit, which stimulates JCV transcription. Together, these findings support the hypothesis that DNA damage inflicted by JCV infection activates the DDR and induces the expression of Rad51, which acts together with NF-KB to promote productive infection. In this proposal, we will test the hypothesis that interplay between the DDR and the NF-KB signaling pathway regulates JCV infection. We propose a detailed experimental analysis of the role of nucleus to cytoplasm NF-KB signaling (AIM 1) and the importance of Rad51 (AIM 2) in JCV infection. The outcome of these studies will provide new insights into the life cycle of JCV, its interactions with the host cell and the pathogenesis of PML. This may lead to the development of new therapeutic avenues for controlling JCV infection and PML, a disease with an escalating public health impact for which there are currently no effective treatment options.